Vibrio cholerae is a gram-negative enteric human pathogen that causes Asiatic cholera, a severe and often lethal form of diarrheal disease. V. cholerae is one of a select number of human pathogens that can cause pandemics, epidemics that span the entire globe. Virulent strains of V. cholerae are routinely isolated locally from brackish waters of the Gulf of Mexico and the Rio Grande River and represent a source of contamination to potable water in the event of an environmental catastrophe such as a hurricane or a bioterrorist attack. The leading cause of the dramatic diarrhea caused by V. cholerae infection is cholera toxin (CT) while the element critical for establishment of infection in the small intestine is a type IV pilus named toxin-coregulated pilus (TCP). Synthesis of CT and TCP is tightly regulated by a signal cascade governed by the transcriptional activators ToxR, TcpP, and ToxT. However, the signals encountered and sensed by V. cholerae leading to CT and TCP production in the small intestine remain unknown. The general aim of this proposal is the identification of yet-unknown virulence factors or signaling cascades contributing to colonization and pathogenesis of V. cholerae utilizing a genome-wide approach that exploits the inability of a defined set of colonization-deficient mutants to produce virulence factors in vitro. The utilization of a genome-wide technology is uniquely suited to V. cholerae because of the requirement of the ToxR/TcpP/ToxT virulence cascade for the production of virulence factors in vivo and in vitro and because the growth conditions that induce expression of virulence genes in the laboratory are known. The central hypothesis of this proposal is that among mRNA transcripts differentially expressed by wild type, virulent V. cholerae compared to colonization defective mutants, grown under in vitro conditions that induce expression of virulence factors, are those of genes contributing to colonization. The list of genes generated from this effort is expected to include open-reading frames whose functions are yet unknown. Coding sequences identified by this strategy will be targeted for in-frame deletions and new mutant strains will be constructed and tested for activation of virulence factors in vitro and colonization of the infant mouse small intestine.